Apparatus and methods for processing biological samples and a reservoir therefor

ABSTRACT

An apparatus for processing at least one biological sample accommodated on at least one carrier member ( 15 ) in a chamber includes, at least one reservoir ( 18 ) able to accommodate a fluid on a surface inside the chamber adjacent to and/or facing a substantial part of the at least one biological sample. The apparatus may comprise a bottom member ( 12 ) arranged to support at least one carrier member ( 15 ) carrying at least one biological sample and a lid ( 14 ) including at least one fluid reservoir ( 18 ). The reservoir filled with water provides humidity to the chamber and impedes drying out of the sample.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 13/016,374,filed Jan. 28, 2011, which is a continuation of application Ser. No.11/031,514, filed Jan. 7, 2005, which claims priority to and thebenefit, under 35 U.S.C. 119(e), of the filing date of U.S. ProvisionalApplication 60/535,615, filed Jan. 8, 2004 and titled “Apparatus andMethods for Processing Biological Samples”, all of which applicationsare incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention pertains to the fields of cytology and histology,molecular biology, biochemistry, immunology, microbiology and cellbiology. In particular, the invention is related to the fields ofmolecular cytogenetics and immunohistochemistry and, even moreparticularly, to a method and an apparatus for processing, treatment, oreven staining of at least one biological sample accommodated on acarrier member, such as a microscopic slide as well as to the control ofthe humidity and temperature during processing. Applications to whichthe present invention may relate especially include in-situhybridization, fluorescent in-situ hybridization, cytology,immunohistochemistry, special staining, and microarrays, as well aspotentially other chemical and biological applications.

BACKGROUND OF THE INVENTION

Histological and cytological techniques have been used to analysebiopsies and other tissue samples, as an aid to medical diagnosis andresearch. Cytology is the study of the structure of all normal andabnormal components of cells and the changes, movements, andtransformations of such components. Cells are studied directly in theliving state or are killed (fixed) and prepared by for example thinlayer preparation systems, embedding, sectioning, and/or staining forinvestigation in bright field, fluorescent or electron microscopes.Histology is the study of groups of specialised cells called tissuesthat are found in most multi-cellular plants and animals. Histologicalinvestigation includes study of tissue and cell death and regenerationand the reaction of tissue and cells to injury, a disease state such ascancer or invading organisms such as HPV (Human Papilloma Virus).Because normal tissue has a characteristic appearance, histologicalexamination is often utilised to identify diseased tissue.

In situ hybridisation (ISH), and Immunohistochemistry (INC) analyses areuseful tools in histological diagnosis and the study of tissuemorphology. In situ hybridisation (ISH), immunocytochemistry andimmunohistochemistry (INC) seek to identify a detectable entity in asample by using specific binding agents capable of binding to thedetectable entity.

A biological sample is in this application to be understood as abiological sample such as histological samples, e.g. tissue and cellspecimens, including cell lines, proteins and synthetic peptides,tissues, cell preparations, blood, bodily fluids, blood smears,metaphase spreads, bone marrow, cytology specimens, thin-layerpreparations, and specifically biological samples on microscope slides.The biological sample may further suitably be selected from histologicalmaterial, including formalin fixed and paraffin embedded material,cytological material, fine needle aspirates, cell smears, exfoliativecytological specimens, touch preparations, bone marrow specimens, sputumsamples, expectorates, oral swabs, laryngeal swabs, vaginal swabs,bronchial aspirates, bronchial lavage, gastric lavage, blood, urine, andbody fluids. Such biological samples may be subjected to varioustreatments. Further, the biological sample may be suitably selected fromnon human sources, including virus and fungus swabs, samples taken frommedical equipment, veterinary samples and food. Also, samples may betaken from hair, organs, sperm and egg cells as well as cell grown invitro. The biological samples are preferably from living or post-mortemtissues of Homo sapiens, but not limited to eukaroytic cells. Examplesinclude detection of prokaryotic organisms, such as Escherichia coli0157 in drinking water.

Slides can be any suitable solid or semi solid support for thebiological sample. In particular, the support may be a microscope slide,a micro array, a membrane, a filter, a polymer slide, a chamber slide, adish, or a Petri dish.

The current invention relates especially—but not exclusively—to in situhybridisation (ISH). In situ hybridisation is a diagnostic method forcharacterization and evaluation of genes, chromosomes, cells, cellaggregates, tissues and other biological samples. In situ hybridisationcan be used to evaluate and characterize the status, geneticabnormalities and other disease states, such as cancer or disease,caused by infectious organisms. Further, it can be used to characterizecells with respect to infectious agents such as, but not limited to,HPV, HIV (Human Immunodefiency Virus) and HCV (Hepatitis C Virus).Molecular genetic events, such as aneuploidy, gene amplification, genedeletion, RNA expression, RNA transportation, RNA location andchromosome translocations, duplications, insertions, or inversions thatare difficult to detect with karyotype analysis, PCR (Polymerase ChainReaction), or LCR (Ligase Chain Reaction) can be characterized by ISH.

The ISH techniques can have the potential to increase the survivalchances of cancer patients by making possible earlier detection ofmalignancy and more accurate prognostic assessments following tumoursurgery. The technique can also be applied to prenatal and postnatalgenetic analysis. Furthermore, the technology can be used forsimultaneous detection of multiple genetic anomalies in an individualcell, and thereby save assay time and limit specimen requirements.

Non limiting examples of diagnostically important ISH assays includedetection of HER-2 (also known as HER-2/neu or c-erbB2), Topo II (breastcarcinoma), telomers, EGFr, C-Myc (breast carcinoma), N-Myc(neuroblastoma); translocation probe pairs for BCR/ABL (chronicmyelogenous leukemia), EWS (Ewing's sarcoma), C-Myc (Burkitt's lymphoma,T cell ALL), acute myeloid leukemia (AML), myeloproliferative disorders(MPD), Myelodysplastic Syndrome (MDS) and centromeric probes forchromosomes 17, 7, 8, 9, 18, X, and Y. Other examples include theanalysis of Epstein-Barr virus, Herpes simplex virus and Humancytomegalo virus, Human papilloma virus, Varizella zoster virus andKappa and Lambda light chain mRNAs. Yet other examples include thedetection and analysis of samples of non-human origin, for example, foodborne parasites and disease causing microbes and viruses. More specificexamples include:

i) the analysis of HER-2/neu, also known as c-erbB2 or HER-2, which is agene that has been shown to play a role in the regulation of cellgrowth. The gene codes for a transmembrane cell surface receptor that isa member of the tyrosine kinase family. HER-2 has been shown to beamplified in human breast, ovarian and other cancers;

ii) the analysis of aneuploidy for chromosomes 3, 7, 17 and loss of the9p21 locus in urine specimens from patients with transitional cellcarcinoma of the bladder;

iii) the detection and quantification of the lipoprotein lipase (LPL)gene located at 8p22 and the C-MYC gene located at the 8q24 region (Twogenetic alterations observed in abnormal cells, such as Prostate cancersamples, are gain of 8q24 and 8p21-22 (LPL) loss of heterozygosity.);

iv) the identification and enumeration of chromosome 8 in cells obtainedfrom bone marrow. An association has been made between trisomy 8 andboth myeloid blast crisis and basophilia (Trisomy 8 is a prevalentgenetic aberration in several specific diseases like Chronic MyelogenousLeukemia (CML), acute myeloid leukemia (AML), and myeloproliferativedisorders (MPD).);

v) the analysis of chromosome aneuploidy like translocations of theimmunoglobulin heavy chain locus (IGH) located at 14q32 and frequentlyobserved in patients with various hematological disorders (These IGHtranslocations result in the upregulation of oncogenes due to thejuxtaposition of IGH enhancers with these oncogenes.);

vi) the identification of inv(16)(p13q22) where the CBFB gene located in16q22 is fused to the MYH11 gene located in 16p13, resulting in achimeric protein product detected in acute myeloid leukemia (AML);

vii) the detection of Human Papilloma Viruses (HPV), which are a groupof small DNA viruses (There are more than 90 HPV types. Persistent HPVinfection may result in cervical cancer, and has also been associatedwith other types of cancer, e.g. colon cancer. HPV types are classifiedaccording to the risk associated with the development of cervicalcancer. Fifteen types are classified as high-risk, and these aredetected in more than 99% of all cervical cancers.).

In summary, the in situ Hybridization (ISH) technique is a useful methodfor the analysis of cells for the occurrence of chromosomes, chromosomefragments, genes and chromosome aberrations like translocations,deletions, amplifications, insertions or inversions associated with anormal condition or a disease. Further, ISH is useful for detection ofinfectious agents as well as change in levels of expression of RNA.

The ISH techniques should be understood to include, for example,fluorescent in situ hybridization (FISH), chromogenic in situhybridization (CISH), Fiber FISH, CGH, chromosome paints and arrays. Inthe following, the ISH technique and procedures are described in greaterdetail. ISH uses nucleic acid probes, designed to bind, or “hybridize,”with the target DNA or RNA of a specimen, usually fixed or adhered to aglass slide. DNA, RNA, PNA, LNA or other nucleic acid probes ofsynthetic or natural origin can be used for the ISH technique. Theprobes are labelled to make identification of the probe-target hybridpossible by use of a fluorescence or bright field microscope. The probeis typically a double or single stranded nucleic acid, such as a DNA orRNA. It is labelled using radioactive labels such as 31P, 33P or 32S, ornon-radioactively, using labels such as digoxigenin, or fluorescentlabels, a great many of which are known in the art. The hybrid is oftenfurther analysed with computer imaging equipment. Since hybridizationoccurs between two complementary strands of DNA, or DNA analogues,labelled probes can be used to detect genetic abnormalities, providingvaluable information about prenatal disorders, cancer, and other geneticor infectious diseases.

Unlike other molecular DNA-based tests, which require cell lysis to freenucleic acids for analysis, ISH allows analysis of DNA in situ, that is,in its native, chromosomal form within the cell or even the nucleus.This feature permits the analysis of chromosomes, genes and otherDNA/RNA molecules of individual cells. For direct-labelled probes, theresults are detected by viewing the samples under a fluorescencemicroscope with appropriate filters. Indirect detection, like CISH,demands additional labelling steps, which typically require streptavidinor antibody-enzyme conjugates or fluorophore-labeled counterparts, andadditional washing steps once the probe is bound to the target.

An exemplified general ISH procedure includes one or several of thefollowing sequential procedural steps:

i) Mounting of the biological sample on slides

ii) Baking at elevated temperatures

iii) Dewaxing or deparaffination if necessary

v) Washing

v) Target retrieval at elevated temperature

vi) Denaturing at elevated temperature

vii) Incubation with blocking reagents

viii) Addition of probe mixture to the sample on the slide.

ix) Placing a coverslip over the sample and the probe mix and sealingwith rubber cement.

x) Hybridization at elevated temperatures.

xi) Washing at elevated temperatures and removal of coverslip

xii) Air drying and counterstaining

xiii) Visualization according to the instruction for FISH or CISH

xiv) Examination and evaluation in a microscope

In more detail, an exemplified FISH protocol for paraffin embeddedtissue sections could include one or several of the following sequentialprocedural steps:

i) Cutting 2-4 micrometer tumour sections from a block

ii) Mounting on slides

iii) Baking at 60° C. for 30 minutes

iv) Deparaffination using xylene

v) Rehydration by immersing in ethanol/water mixtures

vi) Pre treating by washing with an aqueous buffer for 10 minutes at 95°C.

vii) Pepsin digesting for 10 minutes at ambient temperature

viii) Washing repeatedly

ix) Dehydration in a series of cold ethanol/water mixtures

x) Air drying

xi) Addition of 10 microliter fluorescent labelled DNA or PNA probemixture per slide

xii) Sealing with a 22 by 22 mm glass coverslip and rubber cement at theedges

xiii) Denaturing at 82° C. for 5 minutes, directly followed by

xiv) Hybridization over night (18 hours) at 45° C.

xv) Removal of the coverslip

xvi) Stringent washing at 65° C. for 10 minutes

xvii) Washing repeatedly with wash buffer

xviii) Dehydration by immersing in a series of cold ethanol/watermixtures

xix) Air drying

xx) Mounting with 10 microliter anti fade solution with DAPI as counterstain

xxi) Sealing with a coverslide

xxii) Examination and evaluation in a fluorescence microscope

The hybridization mixture is typically a complex mixture of manycomponents. Non-limiting examples of components include formamide,water, triton x-100, tween 20, Tris or Phosphate buffer, EDTA, EGTA,polyvinylpyrrolidine, dextran sulfate, Ficoll, or salmon sperm DNA.

Chromogenic in situ hybridization (CISH) uses labelled probes, which canbe visualized by the use of immunological staining methods similar tothe IHC staining procedures. CISH has some differences compared to FISHtechniques: The genetic aberrations may be viewed within the context oftissue morphology—simultaneous examination of histopathology and ISHresults. Also, the results may be visualized with a standard brightfield microscope, and the chromogenic dye (for example DAB) generated onthe slide is permanent with no or little fading of fluorescent signals.

In addition to ISH, the current invention also relates toimmunohistochemistry and immunocytochemistry. The general exemplifiedformalin fixed paraffin embedded (FFPE) immunohisto chemical (IHC)chromogenic staining procedure may involve the steps of: cutting andtrimming tissue, fixation, dehydration, paraffin infiltration, cuttingin thin sections, mounting onto glass slides, baking, deparaffination,rehydration, antigen retrieval, blocking steps, applying primaryantibody, washing, applying secondary antibody-enzyme conjugate,washing, applying enzyme chromogen substrate, washing, counter staining,cover slipping and microscope examination.

As described above, the sample treatment of the slides is complicated,laborious and uses many different reagents at various temperatures forprolonged periods. It should be understood that under normal conditionsonly small amount of reagents, 200 μl or even less, are applied to thesample. Thus, the reagent and sample are very easily dried out,especially under high temperatures and at low relative humidity. Many ofthe procedural steps in ISH, including the denaturing and thehybridization steps are typically done in a humidity chamber. Thehumidity chamber is a closed or semi closed container in which theslides can be processed and heated. It should be understood that theprocessing temperature as well as the temperature ramp time—that is, thechange of temperature per time unit, is important for both the overallprotocol length and the subsequent visualized result. Furthermore, ithas been observed that the staining result depends strongly on thehumidity during the sample treatment. Also, the morphology can sufferfrom drying out during the treatment. For example, chromosome spreadsare easily ruined due to drying out conditions. During the changes oftemperatures the air above the slides will expand or contract. Thereduction in pressure during lowering of the temperature will draw inair from the outside, which may be less saturated with water compared tothe air above the slide. During heating, air will be pressed out of thespace between the slide and the lid. This air will contain moisture,which will escape from the system. Consequently, due to the plurality offast and repeated changes in temperature, high temperatures forprolonged time and the small space between the slides and the lid,moisture can escape either quickly, or over time, from the system,resulting in a change in the concentration of the reagents applied tothe biological sample and thus a change in the protocol, or even dryingout of the biological sample.

The absolute humidity is defined as the amount of water in a givenvolume of gas. The relative humidity is the ratio between the amount ofwater and the maximum amount of water possible at the given temperatureand pressure. The maximum amount of water per volume, and consequentlythe relative humidity, depends strongly on the temperature, as describedby the Clausius-Clapeyron equation. For example, without addition ofwater in a closed system, 100% relative humidity at 25° C. willcorrespond to 16.3% at 60° C. and 3.7% at 95° C., indicating the strongdependence of temperature. Even a small change of temperature willchange the relative humidity dramatically. For example, a relativehumidity of 100% at 80° C. will correspond to only 66.7% at 90° C. in aclosed system without addition of moisture.

From the discussion above, it should be clear that precise control ofhumidity, heating and cooling is essential for obtaining, for example,consistent ISH results. Therefore, without an efficient humidifyingsystem, heating of the slides can result in fast drying out of thereagents or sample.

DESCRIPTION OF PRIOR ART

In order to prevent drying out or loss of “reagents” of the slides,several different closed humidifying systems are known to be used in thecytogenetic, pathology and research laboratories during for example thecritical steps of denaturation and hybridization.

U.S. Pat. No. 6,555,361 discloses a hybridization chamber that containsa built-in mechanism for saturating the air within the chamber whensealed thereby preventing drying of the liquid sample. The hybridizationchamber is defined by matching top and bottom clam-shell like halvesthat, when brought together, are sealed by an O-ring and clampingdevice. The chamber is equipped with a liquid reservoir, the liquid fromwhich will serve to saturate the volume of air sealed within thehybridization chamber. A saturated atmosphere within the chamberprevents evaporation of the sample. This patent illustrates an interiorchamber sized to receive a glass microscope slide and suggestspositioning a well within the chamber to retain liquid separately fromthe region for holding a liquid sample. Further, it is suggested todispose a microporous membrane material in the chamber and specificallyin the well. The control of temperature and humidity inside the chamberduring rapid warm-up or cool-down periods is not discussed in thispatent document.

Humid boxes or humidified chambers are typically plastic containers witha lid. Water-soaked paper towels are placed in the bottom of the box andexcess water decanted away. Slides in racks can be placed horizontallyor vertically in the box during for example overnight hybridization.Typical “home-made” humid box laboratory equipment includes standardTupperware™ or Rubbermaid™ boxes or standard cake pans with a tightclosing lid. Wet paper tissue is placed in the bottom. A frame or gridis placed over the tissues and the slides placed on the frame or gridbefore the lid is closed. The humid box is placed in a conventional ormicrowave oven or on top of a heating plate during for example thedenaturation or hybridization steps. To further control the humidity andtemperature profile, the humid box can be isolated to limit heat lossand thereby hold the temperature for longer periods.

An insulated box like e.g. the HybBox™ (InSitus BioTechnologies,Albuquerque, N. Mex., USA) made of expanded polystyrene with a base anda lid is an attempt to further control the humidity and temperatureduring for example hybridization. After denaturation of the biologicalsample on slides in an oven, the slides are transferred to the HybBox™,which is tightly closed. After hybridization, the box is opened and theslides further treated.

To further control the temperature profile during general slideprocessing, several temperature-controlled chambers are commerciallyavailable. One example is the Boekel Slide Moat™ (Boekel Scientific,Feasterville, Pa., USA) consisting of a temperature controlled heatingblock. Up to 30 standard microscope slides can be placed horizontally onthe heating block. A glass lid with seals closes over the heating blockand the slides. Placing wet towels on the heating block together withthe slides can give high humidity.

The HYBrite™ Denaturation/Hybridization System (Vysis, AbbottLaboratories, Downers Grove, Ill., USA) is another temperaturecontrolled humid chamber widely used in, especially, ISH laboratories.It consists of a programmable heating plate on which up to 12 microscopeslides can be placed. A lid comes down over the heating plate and slidesand closes the system. On each side of the slide heating plate, wells orchannels can hold water or wet tissues or towels. Humidity is therebysought controlled by the use of wet tissues or towels. Once the slidesare placed in the instrument and the lid is closed, sequentialdenaturation and hybridization steps can be performed automaticallywithout the intervention of the user.

In an attempt to further control the temperature and avoid smalltemperature fluctuations, the TruTemp heating system (Matrix, Hudson,N.H., USA) uses a heated lid in addition to the heated slide block. Theinstrument consists of a programmable heating block on which the slidesare placed. The lid is further heated. Humidification is provided bywater added to wells integrated into the heating block on which theslides rest.

Typically, the user can program the various commercially availabletemperature controlled humid systems with many time-temperatureprotocols from 0 to more than 24 hours and from ambient temperature to100° C.

In yet another attempt to automate the temperature and humidity duringprocessing, automated instruments using so called liquid coverslipsystems (Ventana Medical Systems, Tucson, Az) have been introduced. Thelimiting of drying out of slide-mounted specimens has been sought bycovering the reagents and sample with an immiscible oil. The system onlylimits the evaporation, resulting in loss of a significant part of thereagent volume and is not practical for hybridization in more than 12hours at elevated temperatures.

The instruments eliminate a number of steps and reduce hands-on timerequired during conventional ISH procedures performed by cytogenetic,pathology and research laboratories. Nonetheless, the manual systemsusing ovens and various plastic containers, in general, still give thebest results with regard to both preserved morphology and stainingefficiency. The semi or fully automated humid boxes or chambers have theadvantage of e.g. less hands-on work and ease of use. However, none ofthe semi or fully automatic humid boxes or chambers has succeeded inproviding a performance equivalent to or exceeding the manual methods,with respect to preserved morphology and staining efficiency. Thehumidity control is closely connected to the temperature, as discussedpreviously, but is not easily controlled. Also, no known system hastruly addressed the problem of having both controllable uniformtemperature and uniform and high humidity over the slides for prolongedtime.

In light of the above discussion, there is a need in the art for animproved treatment device for treating biological samples. In summary,the improved sample treatment device should ideally include:programmable temperature control; precise control of heating andcooling; fast change of temperature; independence of the number ofslides treated; high humidity at any relevant temperature; constanthumidity for prolonged periods; and uniform temperature and humidityover the slides. The present invention addresses such a need.

SUMMARY OF THE INVENTION

The present invention provides an apparatus for processing biologicalsamples, the apparatus comprising means for processing at least onebiological sample accommodated on at least one carrier member,characterised in that at least one reservoir able to accommodate a fluidis arranged on a surface adjacent to and/or facing a substantial part ofthe at least one biological sample. The proximity of the fluid reservoirand the sample is important in order to ensure that vapor from thereservoir can be generated at a rate able to maintain a constant highrelative humidity in the chamber formed around the sample by the innersurfaces of the apparatus during a heating period with raisingtemperature.

In a preferred embodiment, the reservoir is arranged above the at leastone sample on the at least one carrier member.

In a preferred embodiment, the apparatus comprises a bottom memberarranged to support at least one carrier member carrying at least onebiological sample and characterised by further comprising a lidincluding at least one fluid reservoir. The preferred position of thefluid reservoir is on the lower surface of the lid. This ensures theoptimal proximity to the samples.

In a preferred embodiment, the lid member is provided with holdingmeans, such as a grid, slots and/or fingers (not shown), supporting theat least one fluid reservoir, arranged to be located above thebiological samples when the lid is closed, thereby covering the bottommember.

In a preferred embodiment, an apparatus according to the invention ischaracterized in that the reservoir is placed less than 5 cm from thecarrier member, and, preferably, less than 1.0 cm from the carriermember, and, yet more preferably, less than 0.50 cm from the carriermember.

Preferably, an apparatus according to the invention is characterized inthat the macroscopic surface area of the reservoir adjacent to and/orfacing the sample on the carrier member is more than 10% of the totalcarrier member area, and, preferably, more than 30% of the total carriermember area and, even more preferably, more than 60%. The extension ofthe reservoir plays an important role in the same way as the proximityby improving the rate by which the relative humidity may be changed aswell as the ability to maintain a prescribed high relative humidityduring a rapid heating period with raising temperature.

Preferably, an apparatus according to the invention is characterized bycomprising heating means for heating the sample on the carrier member.Preferably, the heating means are incorporated in the apparatus.

Preferably, the apparatus is characterized by comprising temperaturecontrolling means controlling the temperature of the carrier member and,thereby, the temperature of the biological sample on the carrier member.Preferably, the apparatus includes temperature-controlling meansenabling an automatic heating of the sample according to instructionsprescribed in a protocol defining the desired processing of the sample.

Preferably, the apparatus is characterized by comprising at least onetemperature sensor connected to the temperature controlling means.

In a preferred embodiment the heating means are heating wires.Alternatively, the heating means may be inductive heating.

Preferably, the temperature controlling means comprises cooling meansfor cooling the sample on the carrier member. The prescribed processingof a biological sample typically involves cooling after a period ofheating. In a preferred embodiment the cooling means are Peltierelements and/or at least one fan.

In a preferred embodiment, the apparatus may comprise heating means forheating the reservoir, and the temperature controlling means may enablecontrol of humidity in the chamber by changing the temperature of thereservoir and/or sample by activating the heating means or the coolingmeans in the bottom member of the apparatus and/or in the lid.

In a preferred embodiment of the apparatus, the heating means forheating the sample on the carrier member and the heating means forheating the reservoir are controlled separately, and may be heated todifferent temperatures, so that the reservoir may become warmer than thesample or vice versa. In this manner, the control of the relativehumidity within the chamber around the sample may be highly improved asa high humidity may be generated fast by raising the temperature of thereservoir, thereby releasing vapor molecules into the atmosphere in thechamber and thereby around the sample. Dependent on the temperature ofthe sample and the reagents on the sample—and such temperature can becontrolled by heating or cooling the support of the sample—the vapor maystay in a balance with the reagents and the sample or may concentrate onthe sample and in the solution comprising the reagents.

Alternatively, if a lower humidity is desired, this may be obtained bylowering the temperature of the reservoir so vapor tends to concentrateon the reservoir and become absorbed by the reservoir so vapor can beextracted from the atmosphere around the sample in case a drying out ofthe sample should be desired.

It is an essential advantage of the new apparatus according to theinvention that a complete control of temperature and humidity in theatmosphere around the biological sample is made possible.

In a preferred embodiment of the apparatus, the reservoir is shaped as asubstantially flat sheet. Preferably, the thickness of the reservoir isless than 1/10 of the length, so that the external surface—also calledthe macroscopic surface—is large compared to the volume. It is essentialthat the reservoir can contain a sufficient volume of water, but it iseven more essential that the surface enabling an exchange of vapor inand out of the reservoir is large enough to enable a rapid release orabsorption of vapor.

In a preferred embodiment of the apparatus, the reservoir is attached toa lid, which, in a closed position, covers the at least one biologicalsample on the carrier member lying on a temperature-controlled plate. Inanother preferred embodiment of the apparatus, the reservoir is the lid,which, in a closed position, covers individual slides with individualtemperature controlled plates. In yet another preferred embodiment ofthe apparatus, the reservoir is the lid, which, in a closed positioncovers several slides lying on a number of temperature-controlledplates.

Preferably, in the apparatus according to the invention, the reservoirmay have a curved surface structure and uneven surfaces, such as acorrugated surface.

Preferably, the fluid in the reservoir is a liquid and the reservoircomprises a medium able to adsorb and/or absorb and desorb and/orrelease the liquid. Preferably, the fluid is substantially pure water.Alternatively, the fluid may be water including additives, such as ananti-microbial agent.

The fluid may comprise formamide, aqueous buffers, alcohols,dimethylformamide, dimethylsulfoxid, N-methyl-pyrolidone, non-aqueousbuffers or complex mixtures containing inorganic salts, detergents, pHbuffers, organic solvents, glycerol, oil and/or water, or mixturesthereof.

Preferably, the reservoir is a device made of a material having a veryhigh internal surface area, such as artificial and natural sponges,comprising a plurality of cavities able to accommodate a fluid.Preferably at least a substantial portion of the surface(s) ishydrophilic.

Preferably, the reservoir is made of a material from the groupcomprising polymeric fiber composites and blends, glass fiber materials,expanded porous polymers, porous ceramics, Rockwool™, wood pulp,cardboard, leather or celluloses based materials.

Preferably, the reservoir is made of a material comprising any of thecompositions from the group comprising polyethylene, polypropylene,polyurethanes, polysulfones, polyvinyl, polyamide, polyisobutylene,siloxane polymers, polyacrylic compositions, ethylene Vinyl Acetate,viscose rayon, polystyrene, macroreticular polystyrene, aliphatic, orphenol-formaldehyde condensate polymers, epoxy, cotton, polysaccharide,modified polysaccharides, wood pulp, calcium carbonate, silica gels,glass fiber, bentonite, perlite and zeolite.

Preferably, the reservoir is made of a material from the groupcomprising manmade or synthetic polymeric bonded, non-bonded, woven orknitted fibers, micro fibers, textiles and tufted textiles. Preferably,the reservoir is made of a material from the group comprising bondedpolyamide, polyester, polyolefines and cellulose acetate fibers.Preferably, the material is made of non-woven and bonded blends ofhydrophilic modified polypropylene and polyethylene micro fibers.Preferably, the material is made of bundles of fibers or other loosematerial retained by a thin wall of film.

Preferably, the reservoir material has a density from 0.050 to 1.5gram/cm³ and, more preferably, from 0.075 to 0.75 gram/cm³. Preferably,the reservoir material has the ability to hold at least a predefinedminimum volume of liquid per carrier member. Preferably, the reservoirmaterial has the ability to hold at least 10 micro-liters (μl) in totalper carrier member, and, more preferably, more than 100 micro-liters(μl) in total per carrier member, such as more than 200 micro-liters(μl) in total per carrier member, and even more than 500 micro-liters(μl) in total per carrier member, and such as more than 1000micro-liters (μl) in total per carrier member.

Preferably, a further reservoir is arranged on top of the lid for fluidcommunication with the absorbing and desorbing reservoir opposite to thebiological sample. The further reservoir can easily be refilled withwater without opening the hybridising chamber, and the further reservoirmay be in fluid communication with the reservoir material though thinchannels in the lid allowing the water to ooze or flow slowly towardsthe reservoir material.

The current invention has solved the problem of control of humidity froman ISH or IHC reaction by having a liquid reservoir very close to andadjacent to, preferably facing, the sample on the slide. Furthermore,the reservoir is designed for fast exchange of humidity between theliquid phase in the reservoir and the vapor phase in the space betweenthe slide and the lid.

The invention also relates to a reservoir. The reservoir according tothe invention is characterised in that the reservoir comprises a mediumcapable of adsorbing and/or absorbing and desorbing and/or releasing theliquid.

Preferably, the reservoir may be shaped as a substantially flat sheet orplate, so that the surface of the reservoir facing the sample is large,preferably larger than the surface of the sample. Preferably, thethickness of the reservoir is less than 1/10 of the length, in order tofit into the chamber surrounding the at least one sample. Typically, thesample support and the cover or lid forming the chamber around thesample can be designed to leave only a little free space between thesample and the cover or lid. By minimising the volume of the chamber, itis easier to limit the evaporation from the sample as well as to controlthe content of the atmosphere in the small chamber.

In one embodiment, the reservoir may have a curved surface structure anduneven surfaces, such as a corrugated surface, in order to increase thesurface area.

Preferably, the macroscopic surface area (external surface area) of thereservoir adjacent to and/or facing the sample on the carrier member ismore than 10% of the total carrier member area, and, more preferably,more than 30% of the total carrier member area and, even morepreferably, more than 60%.

Preferably, the reservoir is a device made of a material having a veryhigh internal surface area, e.g. comprising a plurality of cavities ableto accommodate a fluid, or wherein the material is made of bundles offibers or other loose material retained by a thin wall of film.Preferably, at least a substantial portion of the surface(s) ishydrophilic.

Preferably, the reservoir may be impregnated with an anti microbialagent or other protective agents. Preferably, the type, shape and sizeof the reservoir material are selected to optimise surface properties tomatch with the liquid surface tension.

The reservoir is a device that can contain liquids, e.g. water, locatedabove or adjacent to the slides and the heating plate below the slides.The liquid can be contained in the reservoir over the slides despite thegravitational forces.

The reservoir has the ability to fast adsorb and/or absorb as well asdesorb and/or release liquids. The reservoir is preferably made of amaterial with very high surface area.

The reservoir can be made of a number of different materials,non-limiting examples including polymeric fiber composites and blends,glass fiber materials, expanded porous polymers, porous ceramics,Rockwool™, wood pulp, cardboard, leather or celluloses based materials.

Further non-limiting examples of reservoir materials include materialscontaining polyethylene, polypropylene, polyurethanes, polysulfones,polyvinyl, polyacrylic, ethylene Vinyl Acetate, viscose rayon,polystyrene, macroreticular polystyrene, aliphatic, orphenol-formaldehyde condensate polymers, epoxy, cotton, polysaccharide,modified polysaccharides, wood pulp, calcium carbonate, silica gels,glass fiber, bentonite, perlite or zeolite. Even a grid of thin steelwires may provide a reservoir for a liquid.

Preferable materials include manmade or synthetic polymeric bonded,non-bonded, woven or knitted fibers, micro fibers, textiles or tuftedtextiles. More preferably, the materials are made of bonded polyamide,polyester, polyolefines or cellulose acetate fibers. Even morepreferably, the material is made of non-woven and bonded blends ofhydrophilic modified polypropylene and polyethylene micro fibers.Further, it should be understood that the reservoir material could bemade of bundles of fibers or other loose material retained by a thinwall of film.

The material can be selected to optimise surface energy to match withthe liquid surface tension. The surface properties of the material canbe due to the bulk material or from specific chemical, plasma orirradiation surface treatments. Such treatments are well known to theperson skilled in the art of polymer chemistry.

The high internal surface areas can adsorb and later desorb a widevariety of different liquids depending on the environment in which theyare used. The relative humidity over the sample on the slide is aconsequence of the absorption characteristics of the reservoir materialand the temperature. Because of the variation options, such as the type,surface properties, shape and size of the reservoir material, theporosity, and the pore size, the broad spectrum of requirements ofhumidification action can be fulfilled.

The reservoir should be selected from materials having a density from0.050 to 1.5 gram/cm³, and, preferably, from materials having a densityfrom 0.075 to 0.75 gram/cm³. Experience has proven that such density ofthe preferred porous or fibrous materials provides the desired absorbingand desorbing features.

The reservoir should be selected from materials and geometrical shapeshaving the ability to hold at least a predefined minimum volume ofliquid per carrier member or microscope slide. Preferably, the reservoirhas the ability to hold at least 10 microliters in total per carriermember, and, more preferably, more than 100 microliters in total percarrier member, and, yet more preferably, more than 200 microliters intotal per carrier member, and, even more preferably, more than 500microliters in total per carrier member, and, most preferably, more than1000 microliters in total per carrier member.

The ability to hold the water is essential, as any water drops on thesample would deteriorate the staining of the sample. Also, a high amountof water in the reservoir is important to obtain a high rate of exchangeof humid air in order to maintain the desired humidity above and withinthe sample.

Preferably, the reservoirs are in the form of flat sheets or plates.Preferably, they may have a curved surface structure and uneven surfacessuch as a corrugated surface to optimise the surface area.

Preferably, the reservoir is sufficiently rigid and stable andself-supporting, and does not creep or bend downward. Also, preferably,the reservoir does not markedly swell or change shape during desorptionor adsorption of liquids or due to change in temperature.

Preferably, the reservoir is placed less than 5 cm over the slides. Morepreferably, the reservoir is placed less than 1.0 cm over the slides,and, yet more preferably, less than 0.50 cm over the slides.

It should be understood that the slides and reservoir could be in atilted or vertical or horizontal position. It is the position of thereservoir adjacent to or facing the sample on the slide which isessential. Also, the arrangement of slides and reservoir could be turnedupside down, so that the reservoir will be located below the slide.

The macroscopic surface area (the external surface) of the reservoirsfacing towards the slides should preferably be more than 10% of thetotal slide area. More preferably, the area should be more than 30% ofthe total slide area. Even more preferably, the area should be more than100% of the total slide area. The macroscopic surface area of thereservoirs should be understood as the external area of the reservoirsand not the internal surface area of the fibers or cavities.

In one preferred embodiment, the reservoir is attached to a lid, whichcomes down over the slides lying on a temperature-controlled plate.Thereby, the slides are enclosed in a closed controllable space,preferably provided with temperature sensors controlling the climate. Inanother embodiment, the reservoir is placed between slides and the lid,which comes down over the slides lying on a temperature-controlledplate. In yet another embodiment, the reservoir is the lid, which comesdown over individual slides with individually temperature-controlledplates, or several slides lying on a temperature controlled plate orplates.

It should be understood that the cover or lid could cover one, two orseveral slides on the temperature-controlled plate or the individuallytemperature-controlled plates.

In yet further embodiments, the reservoir as described above may befurther temperature controlled by a heating device above the reservoir.Preferably, one or more sensors are arranged for sensing temperatureabove the slide(s).

If the reservoirs are attached to the lid and are to be changed, it ispreferred to have small handles or perforated taps in the material foreasy manual manipulation.

Preferably, heating from beneath the slide controls the temperature ofthe slide and biological sample. However, it should be understood thatthe reservoir could also be heated. Preferably, this can be done byelectrical heating wires embedded in the reservoir material or from aheating plate in the lid. This will further increase the efficiency ofthe reservoir's ability to humidify the air over the slides, as apre-warmed reservoir will more easily humidify the space over theslides.

It should further be understood that the reservoir could be connected toexternal reservoirs by tubing or other means to allow increasedcapacity. Consequently the reservoir can be easily refilled. Also,different liquids can be added depending on the reaction and theprotocol defining the sample processing.

It should be understood that, in some applications, the temperaturemight be ambient for long periods. That is, the slides may not be heatedto above the ambient temperature. This is of relevance for storage ofslides overnight before or after staining or for expanded incubationswith reagents.

The reservoir can hold water, aqueous buffers, formamide, alcohols,dimethyl-formamide, dimethylsulfoxide, N-methyl-pyrolidone, non-aqueousbuffers or complex mixtures containing inorganic salts, detergents, pHbuffers, organic solvents, glycerol, oil and/or water as well asmixtures of the above-mentioned liquids.

Further, it should be understood that the composition of the liquidmight include an anti microbial agent, an UV or other protective agents.

Further, it should be understood that the composition of the liquid inthe reservoir might not be the same as in the vapor phase i.e. the vaporin the atmosphere in the environment between the reservoir material andthe biological sample and reagents on a carrier. By adjusting thecomposition of the liquid in the reservoir, the composition of thevapors over the slides may be controlled. Specifically, throughadjustment of the temperature of the liquid components in the reservoir,the content in the environmental vapor phase can be influenced.Experience has proven that, by maintaining a high humidity close to 100%during the relevant processing, the water content in the sample will, bythe end of such processing (typically after a heating, and cooling andstoring over night) be about the optimum for obtaining a perfectstaining of the sample.

Further, it should be understood that the current invention especiallyrelates to semi or fully automated instruments. Especially, computercontrolled and programmable automated instruments for handling andprocessing slides will benefit from this invention.

The humidity above slides positioned individually, or in rack orcarrousel arrangements in instruments can be controlled by the currentinvention. In fact, the invention is not limited to any particulararrangement of the slides on a slide platform. The reservoirs can bepositioned in a stationary position adjacent to the slides.

Alternatively, the reservoirs or slides can be moved to be adjacent toeach other when humidity is to be controlled. The reservoir can besingle use, disposable or more permanently used in a semi or fullyautomated instrument.

Of particular relevance is the use of a lid made entirely or partly ofthe reservoir of the invention. The lid may be placed adjacent to andpreferably facing the slide, and the lid controls the humidity, i.e. asufficiently wet lid will provide for almost 100% relative humidity.Preferably, the lid is placed over the slide while the slide ispositioned over a heating device. Reagents or other liquids can be addedto the slide through one or several holes in the lid.

An automated dispensing device can deliver the reagents. Similarly,liquids can be added to the reservoir by automated dispensing devices inthe instrument.

Further, it should be understood that the current invention would alsofunction as a general warmer of microscope slides or other supports, byspecifying a constant time and uniform temperature and humidity.

Another application, which will benefit from the current invention, isISH on arrays. The arrays can contain thousands of spots or dots ofsample. For example, the spots or dots could comprise immobilizedtissue, genetic material, DNA, cDNA or RNA. The processing andvisualization protocols resemble the protocols of more traditional ISH.Similarly, the control of humidity is essential for consistent results.

Applications using flat membranes or gels, like the one used in westernand northern blots and treatment of electrophoresis gels will benefitfrom the highly controlled humidity and temperature of the currentinvention.

The PCR and LCR technique is only with difficulty performed in situ onsamples mounted on slides. One of the problems is the lack ofstandardization with respect to temperature ramp time and uniformhumidity control. The PCR or LCR procedures, which include repeatedchanges of temperature for long periods could benefit from the currentinvention.

Another application, which will benefit from the current invention, isthe implementation of ISH on arrays. The arrays can contain thousands ofspots, dots, sample dots or tissue samples on a single small or largeslide or planar support. The uniformity of treatment over the many dotswith respect to temperature and humidity is particularly important toensure reproducible results.

Also, it should be understood that the current invention could reducethe humidity. The ability of the reservoir to efficiently adsorbmoisture will create a dehumidifying system. As an example, such abilitycould be desirable when the temperature of the slide is decreased, whichimplies that some vapor in the air over the slide will be released aswater, and this water has to be removed from the air over the slide. Byhaving the hydrophilic reservoir, such vapor can be adsorbed on thehydrophilic fibers.

For example, by using a dry reservoir, with no or little liquid present,the high area surface removes the liquid between the space of the slidesand the reservoir. This will result in fast dehydration of the slides.Furthermore, applying heat to the slides will increase the speed andefficiency of the dehydration process. For example, as describedpreviously, the typical ISH protocol includes a dehydration step afterthe stringency wash step. The stringency wash is followed by two washsteps, by which the slides are immersed in a series of baths withincreasing ethanol concentration and left to air-dry, before addition ofmounting medium.

By using a reservoir with the capability to adsorb liquid, the number ofsteps in the process can be reduced. Heat applied to the slides willfurther speed up the process. In summary, an efficient dehumidifyingsystem can reduce the steps and reagents needed for dehydration ofslides.

The invention further relates to a method of processing biologicalsamples wherein at least one biological sample is arranged on a carriermember, for treatment in order to prepare the sample by staining for avisual analysis of the sample, characterised by maintainingsubstantially at least 80% relative humidity above the sample throughthe close presence of a reservoir filled with water. Preferably, themethod is characterized by maintaining relative humidity in theatmosphere above the sample of substantially at least 85% and,preferably, at least 90%, and, more preferably, at least 95% relativehumidity and, most preferably, 99-100% relative humidity through theclose presence of a reservoir, filled with water.

When carrying out the method of processing, it is preferred to supplythe reservoir with water after the arrangement of the samples on thecarrier members. If the lid with the reservoir material comprising thecontent of water is left open for a substantial time, the water may oozedownwards flowing out of the reservoir. Preferably, the lid is closedand positioned in its normal, horizontal position when it containswater—and during the processing of the samples.

Finally, the invention relates to the use of a reservoir in an apparatusfor executing a method of processing biological samples, wherein atleast one biological sample is arranged on a carrier member, fortreatment in order to prepare the sample by staining. Experiences haveindicated that the invention is particularly useful for hybridising asample for performing an analysis in which DNA is the target, for HPV,Her-2, Top2A; for hybridising a sample for performing an analysis inwhich RNA is the target; for HPV; for performing IHC analysis; for p16,Her-family including phosphorylated ER/PR, MIB-1, and for hybridising asample for performing an analysis from the group comprising ISH, HPV,HER 2, HER2FISH, Topo II, telorners, EGFr, C-Myc, Epstein-Barr virus,Herpes simplex virus and Human cytomegalo virus, Chronic MyelogenousLeukemia (CML), acute myeloid leukemia (AML), Chromosome banding andpaints.

BRIEF DESCRIPTION OF THE DRAWINGS

The object and features of the present invention can be more fullyunderstood and better appreciated with reference to the attacheddrawings, which are schematic representations only and not necessarilydrawn to scale, wherein:

FIG. 1 shows a preferred embodiment of an apparatus in accordance withthe present invention with the lid open.

FIG. 2 shows the same apparatus as in FIG. 1 with the lid closed.

FIG. 3 shows a schematic view of an arrangement of carrier members on abottom member of the apparatus of FIG. 1.

FIG. 4 shows a sectional view of the apparatus of FIG. 3 along the linea-a in FIG. 3.

FIG. 5 shows a sectional view of the apparatus in FIG. 3 along the lineb-b of FIG. 3.

FIG. 5A shows a sectional view similar to FIG. 5, but with a heatingplate in the lid.

FIG. 6 shows a sectional view similar to FIG. 5 of an embodiment of anapparatus in accordance with the present invention having an externalreservoir.

FIG. 7 shows the display and keypad of the apparatus of FIGS. 1 and 2.

FIG. 8 shows a single tissue slide on a heating plate, covered by areservoir according to an embodiment in accordance with the invention.

FIGS. 9 and 10 show a manual version of the apparatus of FIGS. 1 and 2.

FIG. 11 shows a slide locator assisting the location of the slides onthe bottom member of an apparatus as shown in FIG. 1, 2, 9, or 10.

FIG. 12 shows a sample on a slide arranged on a heating plate andcovered by a reservoir and a lid according to a method of the presentinvention.

FIGS. 13 and 14 show an apparatus similar to the apparatus shown inFIGS. 9 and 10, but designed for only one slide.

FIG. 15 shows a sectional view of the apparatus of FIGS. 13-14.

FIG. 16 shows an arrangement similar to FIGS. 12-14, including a robotarm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an improved apparatus and methods forprocessing biological samples. The following description is presented toenable one of ordinary skill in the art to make and use the inventionand is provided in the context of a patent application and itsrequirements. Various modifications to the preferred embodiment will bereadily apparent to those skilled in the art and the generic principlesherein may be applied to other embodiments. Although various componentsare discussed in the context of a particular initial design, it shouldbe understood that the various elements can be altered and even replacedor omitted to permit other designs and functionality as appropriate.Thus, the present invention is not intended to be limited to theembodiments shown but is to be accorded the widest scope consistent withthe principles and features described herein. To more particularlyappreciate the features and advantages of preferred apparatuses andmethods in accordance with the present invention, the reader is referredto the appended FIGS. 1-16 in conjunction with the following discussion.It is to be understood that the drawings are diagrammatic and schematicrepresentations only and are neither limiting of the scope of thepresent invention nor necessarily drawn to scale.

FIG. 1 illustrates as an example an embodiment of an apparatus 10according to the present invention. The apparatus comprises a bottommember 12 and a lid member 14. Preferably, the bottom member 12 and thelid member 14 are connected through a hinge, which is not shown. In theclosed position illustrated in FIG. 2 the two members provide a closedor at least semi-closed chamber.

A plurality of biological samples on carrier members 15 may be arrangedon the bottom member 12 e.g. as shown in FIGS. 3 and 11. Typically thesamples may be tissue samples on microscope slides 15. An apparatus ofthis kind is manufactured and sold by StatSpin, MA, US and byDakoCytomation, Denmark A/S.

The bottom member 12 includes a temperature controlled heating plate 16,as illustrated in FIG. 4. The heating plate 16 can be made from heatconducting material such as a metal, e.g. such as copper. Alternativelyit could be a heat-conducting polymer. The heating plate includesheating means (not shown) such as heating wires for electrical heating,as well as sensor means 34 for sensing the temperature. Such temperatureregulation is well known and will not be described in further detailshere. Preferably, also cooling means (e.g. Peltier elements and/orfan(s) blowing air), are provided in order to enable a rampedtemperature profile. The final result of the sample treatment may behighly dependent on an exact optimised temperature profile, requiringthat the temperature can be changed rapidly according to therequirements defined at a protocol for the treatment of the biologicalsamples presently arranged in the apparatus.

Preferably, the lid member 14 is provided with holding means, such as agrid, slots and/or fingers (not shown), supporting two humidity controlstrips 18 (FIGS. 4-6), arranged to be located above the biologicalsamples when the lid 14 is closed, thereby covering the bottom member12, as indicated in FIG. 2. The strips 18 act as water reservoirsensuring a presence of water inside the closed apparatus during thetreatment of the biological samples. The strips may be attached by anyknown kind of attachments or adhering means, or may be integrated intothe lid or cover 14.

In a preferred embodiment, the lid member 14 may be provided withfurther heating and/or cooling means 16 a (FIG. 5A), as well astemperature sensing means. Preferably, a temperature-controlling unit inthe apparatus is arranged to allow for setting the temperature of thelid to a value different from the temperature selected for the heatingmember 16 in the bottom member 12 in order to accelerate a release orabsorption of vapor from the chamber. This could be specificallyrelevant during a rapid heating or cooling phase of the sampleprocessing during which the relative humidity can be difficult tocontrol without this extra heating or cooling of the water reservoir.

In a further embodiment, the lid member 14 may be provided with afurther reservoir 28 (FIG. 6) that allows refilling with liquid 28 aduring the sample processing.

It is essential that the strips 18 have large internal surfaces comparedto their external surfaces as well as to their total volume. Thematerial may be of a kind comprising pores, forming the cavitiesaccommodating the water. It is however presently preferred that thecavities are formed by spaces between randomly located bonded fibers,preferably having hydrophilic properties. The strips or reservoirs 18can be made of a number of different materials, non-limiting examplesinclude polymeric fiber composites and blends, glass fiber materials,expanded porous polymers, porous ceramics, Rockwool™, wood pulp,cardboard, leather or celluloses based materials.

Non-limiting examples of materials for strips or reservoirs 18 includematerials containing polyethylene, polypropylene, polyurethanes,polysulfones, polyvinyl, polyacrylic compositions, ethylene VinylAcetate, viscose rayon, polystyrene, macroreticular polystyrene,aliphatic, or phenol-formaldehyde condensate polymers, epoxy, cotton,polysaccharide, modified polysaccharides, wood pulp, calcium carbonate,silica gels, glass fiber, bentonite, perlite or zeolite. Other preferredmaterials include man-made or synthetic polymeric bonded, non-bonded,woven or knitted fibers, micro fibers, textiles or tufted textiles. Morepreferably the materials are made of bonded polyamide, polyester,polyolefins or cellulose acetate fibers.

In the presently preferred embodiment, the strips 18 are oblong platesmade of non-woven and bonded blends of hydrophilic modifiedpolypropylene and polyethylene micro fibers. Preferably, the materialhas a density from 0.050 to 1.5 gram/cm³, more preferably from 0.075 to0.75 gram/cm³. This composition provides the strips with extremely largeinternal surfaces. The hydrophilic properties enable the internalsurfaces to adhere to tiny little water drops, providing a very largesurface of water versus air, thereby enabling and improving a fastexchange and balancing between the liquid phase and the vapor phase ofthe water.

Preferably, the macroscopic surface area of the strips (reservoir) 18facing towards the carrier member with the sample is more than 10% ofthe total carrier member area, and preferably more than 30% of the totalcarrier member area even more preferably more than 50% of the totalcarrier member area, and, in the most preferred embodiment, more than80% of the total carrier member area.

In a presently preferred embodiment, the strips 18 are about 2 mm thick,about 28 mm wide, and about 250 mm long. This structure provides a largesurface of the strip facing the surface of the sample within a shortdistance from the sample. Preferably, the humidity control strip islocated close to the sample in order to improve the fast exchange andsupply of humid air. Preferably, the strips may hold more than 10microliters in total per slide, more preferably, more than 200microliters in total per slide, and yet more preferably, more than 500microliters in total per slide, and even more preferably, more than 1000microliters in total per slide.

By mounting the control strips on the inner surface of the lid andpreferably directly above the sample carriers the distance from thestrips to the sample is minimized. Typically the distance may be 1 or 2mm or even less, but always greater than zero so a layer of air andvapor separates the strip from the sample. The control strip should notget in touch with the sample.

In a further advantageous embodiment the strips may have a curvedsurface structure and uneven surfaces, such as a corrugated surface.Thereby the external surface comprising openings into the interiorsurfaces becomes large improving a rapid exchange of vapors, morespecifically air and vapor of water providing almost 100% relativehumidity.

In yet a further advantageous embodiment the humidity control strips 18may have been impregnated with an anti microbial agent, an UV agent orother protective agents.

In the presently preferred embodiment the reservoir 28 is located abovethe sample on the carrier member so that the water supply is assistedthrough gravitation.

As explained earlier, a high humidity is essential to the final resultof the staining of the biological samples. The presence of water isessential in order to maintain a high humidity. The treatment of thesamples including several, possibly rapid temperature changesnecessitates a rapid exchange between the liquid phase and the vaporphase of the water in order to ensure maintenance of a high relativehumidity in the atmosphere above the samples. Such high relativehumidity can be maintained through the use of the apparatus according tothe invention incorporating the strips 18.

In the presently preferred embodiment, the strips are made of materialsselected for their hydrophilic properties. However, other fluids mightbe contemplated, and in such cases the strip material must be chosen toco-operate with such fluid, e.g. a formamide. More specifically thetype, shape and size of the reservoir material should be selected tooptimise surface properties to match with the liquid surface tension.

In a preferred embodiment, the apparatus comprises data processing meansas well a data input and output means 20, such as a keyboard or keypadand a display means 22 in FIGS. 1 and 7, or is adapted for communicationwith a computer, such as a PC. Preferably, the data processing means mayreceive input from the temperature sensing means, and should be able toprovide control signals to the heating and/or cooling means.

The computer may be provided with software and instructions enabling anautomatic control of temperature and humidity inside the apparatusaccording to protocols specifying the conditions, e.g. temperatures andtimes, for the treatment of the samples.

In a further embodiment, the lid 14 itself is a sheet of hydrophilicmaterial of a type or material as described previously herein for strips18.

Heating wires may be embedded in the hydrophilic material. Also, thematerial may be bi-layered.

The lid 14 may simply be arranged on top of a heating plate carrying thesample carriers (microscope slides).

The following examples show preferred methods of how to use thepreferred embodiment of an automatic apparatus:

Example A Unit Power Up

After a user assures that the unit is plugged into an appropriateoutlet, the user moves a power switch (not shown) to its “ON” position.The instrument then audibly beeps to announce that the power has beenturned on, a cooling fan and heating (not shown) will start and a MainMenu as shown in Table I is displayed on display means 22, when theheating plate in the instrument has reached a default temperature of 37°C.

TABLE I Run a PGM Edit a PGM Create a PGM

Example B Denaturation and Hybridization Program

After the Main Menu screen is displayed, a cursor on the menu highlightsthe “Run a PGM” line of the menu. The user then presses an “Enter” keyof the input and output means 20 to accept this menu item.

Subsequently, using the arrow keys, the user scrolls through variousprogram numbers or program names. To accept the selection of a program,the user presses the “Enter” button or key of the input and output means20. The display 22 then confirms the PGM number/name and Denaturationand Hybridization times and temperatures, an example of which is shownin Table II. The cursor highlights the “Run PGM” line. The user thenpresses the “Enter” button or key to accept this choice.

TABLE II PGM 01 Her2 82° C.: 05; 45° C. 20:00 Run PGM Main Menu

The display 22 then prompts the user to “Add Slides and Close Lid” asillustrated in Table Ill. Before adding slides, the user inserts twoHumidity Control Strips 18 into the inside slide lid. After stripinsertion, and after adding the slides, the user saturates the strips 18with distilled water or equivalent (approx. 13 mL for dry strips). Thecursor then highlights “Start” line. The user presses the “Enter” buttonor key to run the program.

TABLE III PGM 01 Her2 Add Slides - Close Lid Start Main Menu

To return to the Main Menu, the user moves the cursor to highlight the“Main Menu” line of the display 22 and presses the “Enter” button orkey. The display indicates “heating” and current temperature of theslides. Once the temperature reaches a denaturation set point, thedenaturation time will count down from the set time as shown in TableIV.

TABLE IV PGM 01 Her2 Denat in Process Denat: 82° C. 02:28 Present Temp:82° C.

The apparatus will then automatically cool to the hybridization settemperature once denaturation is completed (Table V).

TABLE V Please Wait Cooling to Hyb 45° C. Present Temp: 58° C.

The hybridization time will then count down from the set time oncetemperature reaches a hybridization set point.

Upon program completion, the unit will audibly beep to alert the userand the display will show “Process Complete” as shown in Table VI. Thehybridization temperature will be maintained until an “End PGM/MainMenu” menu selection is accepted by pressing the “Enter” button of inputand output means 20. Before pressing the “Enter” button, the user mayremove slides for further processing. If the “End PGM/Main Menu”selection is not accepted within the first minute of program completion,the hybridization time will start counting the total time athybridization temperature.

TABLE VI PGM 01 Her2 PROCESS COMPLETE Total Hyb Time 21:05 End PGM/MainMenu

Example C Run a Hybridization Only Program

After the Main Menu screen is displayed, a cursor on the menu highlightsthe “Run a PGM” line of the menu. The user then presses an “Enter” keyof the input and output means 20 to accept this menu item.

Subsequently, using arrow keys, the user scrolls through various programnumbers or program names. To accept the selection of a program, the userpresses the “Enter” button or key of the input and output means 20. Theuser selects a Hybridization Only program and the display 20 thenconfirms the PGM number/name and times and temperatures for aHybridization Only protocol, examples of which are shown in Table VII.The cursor highlights the “Run PGM” line.

TABLE VII PGM 02 EBV Hyb: 55° C. 01:30 Run PGM Main Menu

The user then installs two Humidity Control Strips 18 into the insideslide lid. After strip installation, and after adding the slides, theuser saturates the strips 18 with distilled water or equivalent (approx.13 mL for dry strips). The cursor highlights the “Start” line and theuser then presses the “Enter” key or button to run the program as shownin Table VIII.

TABLE VIII PGM 02 EBV Add Slides - Close Lid Start Main Menu

The instrument will heat slides to the hybridization temperature asindicated in Table VIIIa.

TABLE VIIIa Please Wait Heating to Hyb 55° C. Present Temp: 45° C.

Once hybridization temperature is reached the display changes as shownin table VIIIb and the time will count down from the set time.

TABLE VIIIb PGM 02 EBV Hyb in Process Hyb 55° C. 01:30 Present Temp: 55°C.

Upon program completion, the unit audibly beeps to alert the user andthe display 22 shows the message “Process Complete” (Table IX). TheHybridization temperature will be maintained until the “End PGM/MainMenu” selection is accepted by pressing the “Enter” button. Beforepressing the “Enter” button, the user may remove slides for furtherprocessing. If the “End PGM/Main Menu” selection is not accepted withinthe first minute of program completion, the hybridization time willstart counting the total time at hybridization temperature.

TABLE IX PGM 02 EBV PROCESS COMPLETE Total Hyb Time 02:15 End PGM/MainMenu

Example D Fixed Temperature Program

After the Main Menu screen is displayed, a cursor on the menu highlightsthe “Run a PGM” line of the menu. The user then presses an “Enter” keyof the input and output means 20 to accept this menu item.

Subsequently, using arrow keys, the user scrolls through various programnumbers or program names. To accept the selection of a program, the userpresses the “Enter” button or key of the input and output means 20. Theuser selects a Fixed Temperature program. The display 20 then confirmsthe PGM number/name and the Fixed Temperature (Table X) and the cursorhighlights the “Run PGM” line of the display 22.

TABLE X PGM 03 Appl Fixed: 65° C. Run PGM Main Menu

By pressing the “Enter” button or key of input and output means 20 torun the program the instrument will heat to the fixed temperature asindicated in Table XI.

TABLE XI Please Wait Heating to Fxd: 65° C. Present Temp: 30° C.When the fixed temperature is reached, the display 22 then prompts theuser to “Add Slides and Close Lid”. Before adding slides, the userinstalls two Humidity Control Strips into the inside slide lid. Afterstrip installation, and after adding the slides the user saturates thestrips 18 with distilled water or equivalent (approx. 13 mL for drystrips) and closes the lid. The cursor highlights the “Start” line ondisplay 22 (Table XII). The user then presses the “Enter” button of theinput and output means 20 to continue the program.

TABLE XII PGM 03 Appl Add Slides - Close Lid Start Main Menu

To return to the Main Menu, the user moves the cursor to highlight the“Main Menu” line of display 22 and presses the “Enter” button of inputand output means 20. The display 22 then indicates the presenttemperature of slides as shown in Table XIII and the timer countselapsed time. (Pressing the “Enter” button by the user will reset thetimer to zero).

TABLE XIII PGM 03 Appl Fixed Temp: 65° C. Reset Timer 01:18:10 EndPGM/Main Menu

The user may use the Arrow keys of the input and output means 20 to movethe highlighted display to the “End PGM/Main Menu” line and then pressthe “Enter” button to end the program.

As the above examples (Example A through Example D) indicate, thereservoirs 18 which are the humidity control strips may be useful in ahybridizer. However they can be used in many other apparatuses. FIG. 8and FIGS. 12-16 show a single tissue slide 15 on a heating plate 16covered by a reservoir 18 according to the present invention. Sucharrangement may be incorporated in several types of apparatus forprocessing samples, such as automatic stainers, both of the carouseltype and as well as stainers with robots moving reagents and/or slides.

Also the arrangement shown in FIG. 8 and FIGS. 12-16 may be used in atilted version. Also the reservoir 18 as shown in FIG. 8 may beincorporated into a lid 14 similar to the embodiment shown in FIG. 1,but with only one reservoir and one slide 15. A heating plate 16 a maybe attached or embedded in the lid 14, e.g. as shown in FIG. 5A.

In FIG. 16 a robot arm 30 is shown arranged above the lid 14. The lid 14is provided with a hole 24 providing an inlet for fluid to the reservoir18 and enabling the robot to provide a fluid, such as water or a reagentto the reservoir and/or to the sample. This is in order to emphasizethat the apparatus according to the present invention may be part of anautomatic sample-processing instrument for processing a plurality ofbiological samples.

FIGS. 9 and 10 show a manual version of the apparatus, similar to theapparatus in FIGS. 1 and 2, but without computer assisted control.

FIG. 11 shows a view similar to FIG. 3, here with a slide locator 32assisting the arrangement of 12 slides (A-L) on the bottom member of theapparatus in FIGS. 1 and 2.

The arrangements as shown in the drawings, and, specifically, theprovision of a reservoir, in cooperation with the temperature sensors(not shown) and in cooperation with adequate control units, such as acomputer, allow for a precise control of the climate around tissue on aslide 15. Specifically the hydrophilic adsorbent medium of the reservoirenables better staining results than hereto known when using automaticsample processing equipment.

In the following is presented seven examples taken from a validationtest of the instrument.

In Example 1, the reservoir material was ordinary filter paper, not therecommended micro fiber material. In all other examples, the tests werecarried out using the recommended micro fiber strips called “HybridizerHumidity Control Strips”. These strips were oblong plates made ofnon-woven and bonded blends of hydrophilic modified polypropylene andpolyethylene micro fibers.

Example 1 FISH Validation

This is an example with TOP2A and paper filter strips. The on averageacceptance criteria of TOP2A: Scoring 1.5-3 (signal intensity andspecificity). A score of at least 2 on average or a deviation scorewithin ±0.5 on average from reference is required. Individual outlierscan be excluded due to obvious reasons and if these are reported. Thefirst run with TOP2A on Hybridizer was performed with paper filterstrips (Filter strips), Table 1A. The instrument was tested with twelveslides from the same tissue block and resulted in an average score ofthe TOP2A signal intensities that resemble the signal intensities of themanual reference slides.

The signal intensities of Green signal, Centromer 17 on Hybridizer,score 2.0, did not resemble the intensities of the manual reference,score 3. Centromer signal intensities with a score less than 1.5 wereobserved for two of the twelve slides. The signal intensity of Centromer17 was, however, on average 2, Red signal, HER2 did resemble the manualreferences, and therefore the acceptance criteria were barely fulfilled.The table shows Raw data of TOP2A probes on sections cut from the sameformalin-fixed, paraffin embedded breast cancer tissue block; Performedon a hybridizer instrument with paper filter strips as humidity strips.

TABLE 1A Run No. 1 Position in Signal Signal Slide Hybridizer/ManualIntensity Intensity Tissue No. test Red Green Structure 1 1 2.5 2.5 3 22 2.5 2.5 3 3 3 1.5 1 3 4 4 3 2.5 3 5 5 3 2.5 3 6 6 2.5 2 3 7 7 2.5 3 38 8 2.5 1.5 2.5 9 9 2 1.5 2.5 10 10 3 2.5 3 11 11 2 1 2.5 12 12 2 2 3 13Manual test 2.5 3 2.5 14 Manual test 2 3 2.5  1-12 Mean 2.4 2.0 2.9 Std0.469 0.656 0.226 13-14 Mean 2.3 3.0 2.5

Example 2 Example with TOP2A and DakoCytomation Hybridizer HumidityControl Strips

A run performed on the validation instrument No. 102 confirmed that theacceptance criteria were easily fulfilled if Hybridizer Humidity ControlStrips (0.198 g/cm³) were used instead of paper filter strips.

The instrument test run was as good as the manual procedure,

In conclusion, the Hybridizer passed the acceptance criteria for TOP2A.The scores of the slides were, when Hybridizer Humidity Control Stripswere used, as good as the manual procedures.

The table (Table 1B) shows Raw data of TOP2A probes on sections cut fromthe same formalin-fixed, paraffin embedded breast cancer tissue block,performed on hybridizer instrument with Hybridizer Humidity ControlStrips (3 mm thick, 0.198 g/cm³). Green signal, Centromer 17; Redsignal, HER2.

TABLE 1B Run No. 1 Position in Signal Signal Slide Hybridizer/ManualIntensity Intensity Tissue No. test Red Green Structure 1 1 3 2.5 3 2 23 2.5 3 3 3 3 3 3 4 4 3 3 2.5 5 5 3 2.5 2.5 6 6 3 3 2.5 7 7 3 3 3 8 8 33 3 9 9 3 2.5 3 10 10 3 3 3 11 11 3 3 2.5 12 12 3 3 3 13 Manual test 3 33 1, 4 Manual test 3 3 3 1.5 Manual test 3 2.5 3  1-12 Mean 3.0 2.8 2.8Stdv 0.000 0.246 0.246 13-15 Mean 3.000 2.833 3.000 Stdv 0.0 0.3 0.0

Example 3 HER2

The on average acceptance criteria of HER2: Scoring 1.5-3 (signalintensity and specificity). A score of at least 2 on average or adeviation score within ±0.5 on average from reference is required.Individual outliers can be excluded due to obvious reasons and if theseare reported. The run with HER2 on Hybridizer was performed withHybridizer Humidity Control Strips (0.270 g/cm³). The instrument wastested with tissue sections of different thickness (2 μm to 6 μm) fromthe same formalin-fixed paraffin-embedded tissue block. The run resultedin scores of signal intensities and tissue structures that resembled themanual reference. No score deviation of ±0.5 grade or above on averagewas observed. In conclusion, the Hybridizer passed the acceptancecriteria for HER2. The scores of the slides were as good as the manualprocedures. Table 2 shows raw data of the HER2 Probe; performed onhybridizer instrument with Hybridizer Humidity Control Strips (2 mmthick, 0.270 g/cm³). Green signal, Centromer 17; Red signal, HER2.

TABLE 2 Position in Run No. 1 Thickness Hybridizer/Manual SignalIntensity Signal Intensity Tissue Slide No. of Tissue test Red Greenstructure 1 2 μm 1 3 3 2.5 2 2 3 2.5 2.5 3 3 3 3 2 4 Manual test 3 3 2.55 Manual test 3 3 2.5 6 4 μm 4 3 2.5 2.5 7 5 2.5 2.5 2.5 8 6 2.5 2.5 2.59 Manual test 2 2.5 2.5 10 Manual test 2.5 3 2.5 11 6 μm 7 3 3 3 12 82.5 2 3 13 9 3 3 3 14 Manual test 2.5 3 2.5 15 Manual test 2.5 3 3 1, 2,3 2 μm Mean 3.0 2.8 2.3 6, 7, 8 4 μm Mean 2.7 2.5 2.5 11, 12, 13 6 μmMean 2.8 2.7 3.0 Manual 4, 5 2 μm Mean 3.0 3.0 2.5 Manual 9, 10 4 μmMean 2.3 2.8 2.5 Manual 14, 15 6 μm Mean 2.5 3.0 2.8

Example 4 MLL and ETV6

The on average acceptance criteria of MLL and ETV6: Scoring 1.5-3(signal intensity and specificity). Score deviation of ±0.5 on averagefrom reference is allowed. Individual outliers can be excluded due toobvious reasons and if these are reported.

The run on Hybridizer was performed with Hybridizer Humidity ControlStrips (0.270 g/cm³). The instrument was tested with sample specimensfrom the same lot of metaphase spreads. The run resulted in betterscores of the MLL and ETV6 signal intensities than observed with themanual references. The structure of the cells resembled the manualreferences. In conclusion, the Hybridizer passed the acceptance criteriafor MLL and ETV6. The scores of the slides were better than the manualprocedures. The scores obtained on Hybridizer were, though, for bothprobes more than 0.5 grade higher in signal than the manual references,These scores are above the deviations described in the acceptancecriteria, but still acceptable.

Table 3 shows raw data of translocation probes, MLL and ETV6, onmetaphase spreads, performed on hybridizer instrument No. 25 withHybridizer Humidity Control Strips (2 mm thick, 0.270 g/cm³).

TABLE 3 Run in Hybridizer No. 25 Position Signal in Structure of SlideProbe in Hy- inter- Signal in inter- and Com- No. mix bridizer phasesmetaphases metaphases ments 1 ETV6 4 3 3 2 — 2 8 3 3 2 — 3 3 2.5 2.5 2 —4 Manual 2 2 2 — 5 test 2 2 2 — 6 MLL 5 2.5 3 2 — 7 6 2.5 2.5 2 — 8 12.5 2.5 2.5 — 9 Manual 1 2 2.5 — 10 test 2 2 2 — Signal of inter- andMethod metaphases Structure Hybridizer 1-3 2.83 ± 0.26 2 ± 0 Hybridizer6-8 2.58 ± 0.20  2.2 ± 0.29 Manual 4-5 2.0 ± 0  2 Manual 9-10 1.75 ±0.5  2.25

Example 5

This example relates to CISH validation of HPV on Formalin-fixedparaffin-embedded tissue blocks. The on average acceptance criteria ofHPV on cells: 2.5-4 signal; 0 negative control; 0-1 background; ±0.25grade divergence from manual staining (for individual slides).

The run with HPV probes on Hybridizer was performed with HybridizerHumidity Control Strips. The signal intensities fully resembled those ofthe manual references. No score deviation was observed. The backgroundlevels appeared to be lower with Hybridizer than with the manual method.

In conclusion, the scores of signal intensities of the slides were asgood as the manual procedure, when the hybridisation was performed withthe humidity control strips

Table 4 Raw data of HPV Probe on Tissue.

The test ran on a Hybridizer with Hybridizer Humidity Control Strips.

TABLE 4 Slide Block Method number No. Signal Background Hybridizer 1 2363 0.25 Hybridizer 2 340 3 0.25-0.5 Hybridizer 6 340 3 0.25 Hybridizer 7236 3 0.5 Hybridizer 11 236 3 0.5 Hybridizer 12 340 3 0.75 Manual 13 2363 0.25 Manual 14 236 3 1 Manual 15 340 3 0.75 Manual 16 340 3 0.5 MethodSignal Background Hybridizer 3 ± 0 0.42-0.46 ± 0.19-0.20 Slide 1, 2, 6,7, 11, 12 Manual 3 ± 0 0.63 ± 0.32 Slide 13-16

Example 6 Telomere

The on average acceptance criteria of Telomere: Scoring 1.5-3 (signalintensity and specificity). Score deviation off 0.5 on average fromreference is allowed. Individual outliers can be excluded due to obviousreasons and if these are reported.

The run on Hybridizer was performed with Hybridizer Humidity ControlStrips (0.22-25 g/cm³). The validation instrument was tested with samplespecimens from two different lots of metaphase spreads. The run resultedin scores of signal intensities and tissue structures that resembled themanual reference for both FISH (K 5325) and Cy3 (K 5326) labelledTelomere probes. No score deviation above ±0.5 grade on average wasobserved. The structure of the cells resembled the manual references.

In conclusion, the Hybridizer passed the acceptance criteria forTelomere. The scores of the slides were as good as the manualprocedures.

Table 5: Raw Data of Telomere Probes, on Two Different MetaphaseSpreads.

Performed on hybridizer instrument with Hybridizer Humidity ControlStrips (0.22-0.25 g/cm³).

TABLE 5 Average Average Metaphase Position in Signal signal signal SlideNo. preparation Probe Hybridizer intensity Background intensitybackground 1 080903- Telomere/ 1 3 0 3 ± 0 0 ± 0 2 MEM FITC 2 3 0 3 3 30 4 Manual 3 0 3 0 5 test 3 0 6 221203- 4 3 0.5 2.67 ± 0.29 0.5 ± 0  7MEM 5 2.5 0.5 8 6 2.5 0.5 9 Manual 3 0 3 0 10 test 3 0 11 080903-Telomere/ 7 3 0 3 ± 0 0 ± 0 12 MEM Cy3 8 3 0 13 9 3 0 14 Manual 3 0 3 015 test 3 0 16 221203- 10  3 0 3 ± 0 0 ± 0 17 MEM 11  3 0 18 12  3 0 19Manual 3 0 3 0 20 test 3 0

Example 7 EBER (EBV)

The on average acceptance criteria of EBER: Scoring 1.5-3 (signalintensity and specificity). Score deviation of ±0.5 on average fromreference is allowed. Individual outliers can be excluded due to obviousreasons and if these are reported.

The run on Hybridizer was performed with Hybridizer Humidity ControlStrips (0.22-25 g/cm3). The run resulted in scores of signal intensitiesthat resembled the manual reference. No score deviation of ±0.5 grade orabove on average was observed. The background appeared to be lower withHybridizer than with the manual method.

In conclusion, the Hybridizer passed the acceptance criteria for EBER.The scores of the slides were as good as the manual procedures.

Table 6: Raw Data of EBER Probes on Two EBV-Positive Tissue.

Performed on 1-lybridizer instrument with Hybridizer Humidity ControlStrips (0.22-0.25 g/cm³).

TABLE 6 Slide Position in Signal No. Tissue Hybridizer Probe mixintensity Background 1A A 1 EBER Y5200 2 0 1B Neg. control 0 0 2A 2 EBERY5200 2.5 0.5 2B Neg. control 0 0 3A B 3 EBER Y5200 2 0.5 3B Neg.control 0 0 4A 4 EBER Y5200 2 0 4B Neg. control 0 0 5A A Manual testEBER Y5200 2.5 0.5 5B Neg. control 0 0 6A EBER Y5200 2.5 1 6B Neg.control 0 0.5 7A B EBER Y5200 2.5 0.1 7B Neg. control 0 0.5 8A EBERY5200 2 0 8B Neg. control 0 0.5 Method Signal intensity BackgroundHybridizer EBER 1A-4A 2.13 ± 0.25 0.25 ± 0.28 Hybridizer neg. control1B-4B 0 ± 0 0 ± 0 Manual EBER 5A-8A 2.38 ± 0.25  0.4 ± 0.45 Manual neg.control 5B-8B 0 ± 0 0.38 ± 0.25

LIST OF REFERENCE NUMBERS

The following is a list of reference numbers used in the accompanyingdrawings and referred to in this specification: 10—apparatus,Hybridizer; 12—bottom member; 14—lid member; 15—carrier members, whichmay be microscope slides; 16—temperature-controlled heating plate; 16a—heating plate in lid 14; 18—humidity control strips or reservoir;20—data input and output means including a display and key pad;22—display; 24—hole; 28—further reservoir for refilling the reservoir18; 28 a—liquid within reservoir 28; 30—robot arm; 32—slide sorter.

An improved apparatus and methods for processing biological samples anda reservoir therefore have been disclosed. Although the presentinvention has been described in accordance with the embodiments shownand discussed, one of ordinary skill in the art will readily recognizethat there could be variations to the embodiments and those variationswould be within the spirit and scope of the present invention. Forinstance, although the preferred embodiment of the present invention isdescribed in the context of a Hybridizer for 12 slides, it will beappreciated that the teachings of the present invention are applicableto any number of slides that are processed in any number of chambersequipped with any system for controlling temperature and humidity, e.g.,in automated sample processing equipment comprising a plurality ofheater plates, each of them being arranged to carry a single microscopeslide with tissue. Also, even though all figures show the reservoirabove the slide on the heater plate in the bottom part, it must beunderstood that the chamber might be turned upside down so that thereservoir would be arranged below the slide. Accordingly, manymodifications may be made by one of ordinary skill in the art withoutdeparting from the spirit and scope of the invention, which is definedby the appended claims.

The invention claimed is:
 1. An apparatus adapted for nucleic aciddenaturization and hybridization of at least one biological sample on atleast one carrier member in a chamber, said apparatus comprising: abottom member supporting said at least one carrier member; a hydrophilicporous solid reservoir configured to sorb and desorb water and watervapor; a lid for the chamber, detachable from the bottom member, the lidand the bottom member forming a closed chamber when the lid ispositioned atop the bottom member, wherein the lid is configured toposition the reservoir inside the closed chamber directly above the atleast one biological sample such that the reservoir does not touch saidat least one biological sample, when said lid is positioned atop thebottom member to form the closed chamber; and a sample temperaturecontrolling device below said at least one carrier member controllingthe temperature of said at least one carrier member and said at leastone biological sample, wherein said reservoir either sorbs or desorbswater and water vapor so as to control relative humidity within saidchamber at above 85% relative humidity, when said lid is positioned atopthe bottom member to form the closed chamber.
 2. The apparatus of claim1, wherein said lid may be removed from the bottom member to access saidat least one biological sample and said reservoir.
 3. The apparatus ofclaim 1, wherein said reservoir is less than 5 cm from said at least onecarrier member.
 4. The apparatus of claim 1, wherein said reservoir isless than 1.0 cm from said at least one carrier member.
 5. The apparatusof claim 1, wherein said reservoir is less than 0.5 cm from said atleast one carrier member.
 6. The apparatus of claim 1, wherein amacroscopic surface area of said reservoir is greater than 10% of thetotal area of said at least one carrier member.
 7. The apparatus ofclaim 1, wherein said sample temperature controlling device furthercomprises at least one element selected from the group consisting of atemperature sensor, heating wires, an inductive heater, and a coolingdevice for cooling the at least one biological sample.
 8. The apparatusof claim 1, wherein relative humidity in the closed chamber iscontrolled by desorption of water and water vapor from said reservoirduring heating of said at least one biological sample or absorption ofwater and water vapor from said closed chamber during cooling of said atleast one biological sample.
 9. The apparatus of claim 1, wherein saidreservoir is shaped as a substantially flat sheet.
 10. The apparatus ofclaim 9, wherein the thickness of said reservoir is less than 10% of thelength of said reservoir.
 11. The apparatus of claim 1, wherein saidwater further comprises at least one additive, the at least one additiveselected from the group consisting of an anti-microbial agent, aninorganic salt, a detergent and an organic solvent.
 12. The apparatus ofclaim 1, wherein said reservoir is made of a material from the groupconsisting of polymeric fiber composites, polymeric fiber blends, glassfiber materials, expanded porous polymers, porous ceramics, wood pulp,cardboard, leather and cellulose-based materials, or is made of amaterial comprising a composition chosen from the group consisting ofpolyethylene, polypropylene, polyurethanes, polysulfones, polyvinyl,polyacrylics, ethylene Vinyl Acetate, viscose rayon, polystyrene,macroreticular polystyrene, aliphatic polymers, phenol-formaldehydecondensate polymers, epoxy, cotton, polysaccharide, modifiedpolysaccharides, wood pulp, calcium carbonate, silica gels, glass fiber,bentonite, perlite and zeolite.
 13. The apparatus of claim 1, whereinsaid reservoir is made of a material comprising bundles of fibers orloose material chosen from the group consisting of polymeric bondedfibers, non-bonded fibers, woven fibers, knitted fibers, micro fibers,textiles, tufted textiles, bonded polyamide fibers, polyester fibers,polyolefin fibers, cellulose acetate fibers, and non-woven and bondedblends of hydrophilic modified polypropylene and polyethylene microfibers, wherein the bundles of fibers or loose material are retained bya thin wall of film.
 14. The apparatus of claim 1, wherein saidreservoir is impregnated with an anti microbial agent or otherprotective agent.
 15. The apparatus of claim 1, wherein the type, shapeand size of material of which said reservoir is comprised is selected tooptimize surface properties of said material with regard to a surfacetension of said water.
 16. The apparatus of claim 1, wherein material ofwhich said reservoir is comprised has a density from 0.050 to 1.5gram/cm³.
 17. The apparatus claim 1, wherein said reservoir has theability to hold at least a predefined minimum volume of liquid percarrier member, the predefined minimum volume being chosen from thegroup consisting of 10 micro-liters, 100 micro-liters, 200 micro-liters,500 micro-liters, and 1000 micro-liters.
 18. The apparatus of claim 1,further comprising a second reservoir outside the closed chamber.
 19. Amethod of processing biological samples, wherein at least one biologicalsample is arranged on a carrier member, for treatment to prepare thesample by staining, the method comprising the steps of: providing abottom member for supporting said carrier member; positioning a lid,detachable from the bottom member, above the carrier member to form aclosed chamber with the bottom member, wherein positioning the lid abovethe carrier member also positions a hydrophilic porous solid reservoirconfigured to sorb and desorb water and water vapor directly above theat least one biological sample such that the reservoir does not touchsaid at least one biological sample; controlling the temperature of saidcarrier member and said at least one biological sample with a sampletemperature controlling device positioned below said carrier member; andmaintaining at least 85% relative humidity inside the chamber throughthe sorption or desorption of water and water vapor.